Microfluidic devices have shown utility in biochemical analysis by increasing throughput and adding functionality to existing macroscale counterparts. However, “microfluidics” has only recently gained momentum in biological applications, especially in those applications involving cell culture. It is thought that scaling down to the cellular scale (micrometers) will provide more biologically relevant outputs by allowing the cells to communicate and behave more like they would in vivo. Convection can be tightly controlled in microscale devices thereby leaving diffusion as the dominant mode of mass transport. With diffusion as the dominant mode of mass transport, the accumulation of cell secretions is promoted. In addition, the accumulation of soluble factors is further promoted by the increased cell volume ratios of microscale devices (i.e., more cells per microliter (μL) of culture media). As such, microscale culture can offer a more sensitive environment for autocrine and paracrine cell signaling. This increased sensitivity, in turn, allows one to detect biological behavior that is undetectable in macroscale analogs.
One of the main strengths of microscale techniques for biology is the reduced volumes of reagents needed per assay. This benefit is typically discussed as being a source of significant and direct cost savings per endpoint. In the context of cell-based assays, more endpoints can be obtained per cell sample, a fact which is particularly important point in the area of primary cell analysis. Further, by obtaining more endpoints per cell sample, the statistical relevance of results can be directly impacted, specifically in analyses that involve rare cell types or samples with small cell numbers. In the area of clinical diagnostics and monitoring, biopsies and tissue sampling are being pushed to be less invasive. As a result, these procedures typically result in smaller and smaller cell samples. Various types of liquid biopsies often fall into this category such as blood samples from patients with lung, prostate, or breast cancer for analysis of circulating tumor cells (CTCs) or the fluid from bronchoaveolar lavages to diagnose lung cancer. Depending upon the extent of disease and volume of fluid sampled, these types of liquid biopsies may only procure hundreds or thousands of the cell type desired for analysis. With such low numbers of cells, it can be difficult to achieve a robust readout and perform replicates using macroscale techniques. Hence, it can be appreciated that it would be highly desirable to extend the functionality of microfluidic devices to more easily accommodate small numbers of cells and to meet this growing need.
Although it is not often discussed, interfacing limited tissue samples collected using macroscale techniques with microscale devices can pose significant challenges. The first challenge is to seed or place the cells in the device. For many types of microfluidic devices, this often involves the use of tubes and syringe pumps that can act as a barrier for widespread use due to the specialized equipment and expertise required.
A second challenge relates to sample pre-processing. As mentioned previously, microculture devices generally use much higher cell:volume ratios (˜5-10 times). For this reason, the densities of cell suspensions are increased accordingly before seeding. However, when the number of cells is extremely small, the corresponding volume of cell suspension for reaching the desired cell density can be too small for typical macroscale prep methods. For this reason, basic centrifugation for pre-concentration has a practical limit in microculture applications using rare cells. For example, if there are less than approximately 50,000 target cells total, centrifugation may be inadequate to supply a high enough cell density for downstream culture or assays of cell function.
Therefore, it is a primary object and feature of the present invention to provide a method of a collecting particles from a sample fluid.
It is a further object and feature of the present invention to provide a method of a collecting particles from a sample fluid that is simpler and less expensive than prior art methods.
It is a further object and feature of the present invention to provide a method of a collecting particles from a sample fluid wherein the density of the particles in the sample fluid is extremely small.
In accordance with the present invention, a method is provided for collecting particles from a sample fluid containing the particles. The method includes the step of providing a microfluidic device having an input channel, an output channel and a collection region. The input channel has an input end and an output end. The output channel has an input end and an output end. The collection region interconnects the output end of the input channel and the input end of the output channel. The sample fluid is flowed through the input channel and the output channel at a first velocity and through the collection region at a second velocity less than the first velocity such that the particles collect in therein.
The method may include the additional steps of filling the input channel, the output channel and the collection region with a fluid. A reservoir drop is deposited on the output end of the output channel and a first pumping drop of the sample fluid is deposited on the input end of the input channel such that the first pumping drop flows through the input channel, the collection region and output channel to the reservoir drop. The reservoir drop is of sufficient dimension to overlap the output end of the output channel and exert an output pressure on the fluid at the output end of the output channel. The first pumping drop exerts an input pressure on the fluid at the input end of the input channel that is greater than the output pressure on the fluid at the output end of the output channel. Thereafter, additional drops of the sample fluid may be sequentially deposited on the input end of the input channel after the first pumping drop flows into the input channel. As a result, a pressure gradient is generated between the fluid at the input end of the input channel and the fluid at the output end of the output channel such that the fluid flows towards the output end of the output channel.
The input channel, the output channel and the collection region have cross-sectional areas. The cross-sectional areas of the input channel and the output channel are less than the cross-sectional area of the collection region. In addition, the input channel, the output channel and the collection region have resistances to flow. The resistances to flow of the input channel and the output channel are greater than the resistance to flow of the collection region.
It is contemplated for the input channel to be a first input channel and the output channel to be a first output channel. The microfluidic device may also include a second input channel and a second output channel. The second input channel has an input end and an output end interconnected to the collection region. The second output channel has an input end connected to the collection region and an output end. The sample fluid also flows through the input channel and the output channel at a first velocity and through the collection region at a second velocity less than the first velocity such that the particles collect in therein.
The output end of the first output channel and the output end of the second output channel may be in fluid communication. The input ends of the first and second input channels may extend radially from a common input port. The collection region may have a generally circular configuration.
In accordance with the present invention, a method is provided for collecting a concentration of particles from a sample fluid containing the particles. The method comprises the steps of: sequentially passing the sample fluid through an input channel having a first cross-sectional area; a collection region having a second cross-sectional area; and an output channel having third cross-sectional area. The cross-sectional area of the collection region is greater than the cross-sectional areas of the input channel and of the output channel. As such, the flow velocities are influenced to allow particles to collect in the collection region.
The input channel has an input end and an output end. The output channel has an input end and an output end. The collection region interconnects the output end of the input channel and the input end of the output channel. The input channel, the output channel and the collection region are filled with a fluid. A reservoir drop is deposited on the output end of the output channel and a first pumping drop of the sample fluid is deposited on the input end of the input channel such that the first pumping drop flows through the input channel, the collection region and output channel to the reservoir drop. The reservoir drop is of sufficient dimension to overlap the output end of the output channel and exert an output pressure on the fluid at the output end of the output channel. The first pumping drop exerts an input pressure on the fluid at the input end of the input channel that is greater than the output pressure on the fluid at the output end of the output channel. Thereafter, a plurality of pumping drops are sequentially deposited at the input end of the input channel after the first pumping drop flows into the first channel. As described, a pressure gradient is created between the fluid at the input end of the input channel and the fluid at the output end of the output channel such that the fluid flows towards the output end of the output channel. The input channel, the output channel and the collection region have resistances to flow. The resistances flow of the input channel and the output channel is greater than the resistance to flow of the collection region.
It is contemplated for the input channel to be a first input channel and the output channel to be a first output channel. As such, the sample fluid is sequentially passed through a second input channel having a cross-sectional area, the collection region, and a second output channel having a cross-sectional area. The first output channel and the second output channel may be in fluid communication. In addition, the first and second input channels may extend radially from a common input port and the collection region may have a generally circular configuration.
In accordance with a further aspect of the present invention, a method is provided for collecting particles from a sample fluid containing the particles. The sample fluid flows through an input channel at a first flow velocity and through a collection region at a second flow velocity less than the first flow velocity such at least a portion of particles in the sample fluid settle in the collection region.
The sample fluid may also be flowed through an output channel downstream of the collection region at a third flow velocity greater than the second flow velocity. The input channel, the collection region and the output channel have resistances to flow. The resistance to flow of the collection region is less than the resistance to flow of the input channel and the output channel. The input channel has an input end and an output end. The output channel has an input end and an output end. The collection region interconnects the output end of the input channel and the input end of the output channel. Thereafter, the input channel, the output channel and the collection region are filled with a fluid. A reservoir drop is deposited on the output end of the output channel and a first pumping drop of the sample fluid is deposited on the input end of the input channel such that the first pumping drop flows through the input channel, the collection region and output channel to the reservoir drop. The reservoir drop is of sufficient dimension to overlap the output end of the output channel and exert an output pressure on the fluid at the output end of the output channel. The first pumping drop exerts an input pressure on the fluid at the input end of the input channel that is greater than the output pressure on the fluid at the output end of the output channel. A plurality of pumping drops may be sequentially deposited at the input end of the input channel after the first pumping drop flows into the first channel. As described, a pressure gradient is generated between the fluid at the input end of the input channel and the fluid at the output end of the output channel such that the fluid flows towards the output end of the output channel.
The input channel, the output channel and the collection region have cross-sectional areas. The cross-sectional areas of the input channel and the output channel are less than the cross-sectional area of the collection region. The input channel may be a first input channel and the output channel may be a first output channel. The sample fluid may be flowed through a second input channel at a fourth flow velocity and through the collection region at the second flow velocity less than the fourth flow velocity such at least a portion of particles in the sample fluid settle in the collection region. In addition, the sample fluid may flow through a second output channel downstream of the collection region at a fifth flow velocity greater than the second flow velocity.
The first output channel and the second output channel may be in fluid communication. The first and second input channels may extend radially from a common input port. The collection region may have a generally circular configuration.